Scytalone dehydrogenase gene showing tolerance to agricultural pesticide

ABSTRACT

The present invention provides a gene that can be used extensively in studies relating to resistant rice blast fungi. The gene codes for either one of the following proteins (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:2; or (b) a protein consisting of an amino acid sequence shown in SEQ ID NO:2 by deletion substitution or addition of one or more amino acids, which exhibits scytalone dehydratase activity in the presence of a scytalone dehydratase inhibitor.

FIELD OF THE INVENTION

The present invention relates to a gene coding for scytalone dehydratasefrom a rice blast fungus, which is known as a pathogenic fungus for riceblast.

BACKGROUND ART

Rice blast caused by rice blast fungi (pyricularia oryzae, Magnaporthegrisea) is recognized in most countries where rice is cultivated. Inparticular, in regions having climates of high temperature and highhumidity (e.g., Japan), rice blast is one of the most serious diseasesin agricultural industry. For high yield rice cultivation, prevention ofand disinfestation for rice blast are essential. Recently, as analternative to agents with treatment effects, box-treatment agentshaving preventive effects are used for reducing the labor of farmers inprevention and disinfestation regarding rice blast fungi. Examples ofsuch agents include scytalone dehydratase (hereinafter, simply referredto as “SCDH”) inhibitors as typified by carpropamid((1RS,3SR)-2,2-dichloro-N-((R)-1-(4-chlorophenyl)ethyl)-1-ethyl-3-methylcyclopropanecarboxamide))(Kurahashi et al., J. Pestic. Sci, 23, 22-28, 1998; Motoyama et al., J.Pestic. Sci, 23, 58-61, 1998). SCDH is an enzyme that catalyzes thedehydration reaction from scytalone to 1,3,8-trihydroxynaphtalene(hereinafter, simply referred to as “1,3,8-THN”) in melanin biosynthesispathways.

When a rice blast fungus ruptures and invades a cuticular membrane of arice leaf surface, the concentration of glycerol in the appressorium, aninfection-specific organ, increases up to 80 atm. In order to enclosethe glycerol within the appressorium, the melanin layer of the cell wallis essential (Kamakura et al., KASEAA, 39, 340-347, 2001). Inhibition ofmelanin biosynthesis prevents formation of the appressorium. Thus, SCDHinhibitors do not have a direct fungicidal action, but rather arenon-fungicidal agents that exhibit prevention and disinfestationactivities by suppressing pathogenicity.

An SCDH gene from a filamentous fungus was first elucidated withPyricularia oryzae. The nucleotide sequence of this gene was notavailable to the public and only the three-dimensional structure of theSCDH protein was reported (Landquist et al., Structure, 2, 937-944,1994). Thereafter, an SCDH gene from Colletorichum lagenarium (Kubo etal., Appl. Environment. Microbiol, 62, 4340-4344, 1996; Accession no.D86079), followed by SCDH genes from Aspergillus fumigatus (Tsai et al.,Mol. Microbiol, 26, 175-183, 1997; Accession no. U95042), Pyriculariaoryzae (Motoyama et al., Biosci. Biotech. Biochem, 62, 564-566, 1998;Accession no. AB004741) and Ophiostoma floccosum (Wang et al., Accessionno. AF316575) were reported. A three-dimensional structure of an SCDHprotein bound to carpropamid has also been reported (Nakasako et al.,Biochemistry, 37, 9931-9939, 1998; Wawrzak et al., Proteins: Struct.Func. Genet, 35, 425-439, 1999).

DISCLOSURE OF INVENTION

Recently, rice blast fungi with decreased sensitivity to SCDH inhibitorssuch as carpropamid (hereinafter, referred to as “resistant rice blastfungi”) have been discovered. As described above, since the SCDHinhibitors such as carpropamid are very important agents in ricecultivation, it is of the utmost concern to investigate sensitivitydeterminant factors in resistant rice blast fungi and to discovereffective methods of prevention and disinfestation for the resistantrice blast fungi in order to maintain stable rice cultivation.

However, studies concerning resistant rice blast fungi, such aselucidation of the sensitivity determinants in the resistant rice blastfungi or localization of habitats of the resistant rice blast fungi havehardly been made at present.

In order to achieve the above-described objective, the present inventorhas undertaken intensive research and succeeded in clarifying thesensitivity determinants in the resistant rice blast fungi, therebycompleting the present invention.

Thus, the present invention encompasses the following.

(1) A gene coding for either one of the following proteins (a) or (b):

(a) a protein consisting of the amino acid sequence shown in SEQ IDNO:2; or

(b) a protein consisting of an amino acid sequence shown in SEQ ID NO:2by deletion substitution or addition of one or more amino acids, whichexhibits scytalone dehydratase activity in the presence of a scytalonedehydratase inhibitor.

(2) A gene according to (1), wherein the scytalone dehydratase inhibitorinhibits dehydration reaction from scytalone to1,3,8-trihydroxynaphtalene in a melanin biosynthesis pathway.

(3) A gene according to (1), wherein the scytalone dehydratase inhibitoris carpropamid.

(4) A scytalone dehydratase encoded by the gene of (1).

(5) A recombinant vector comprising the gene of (1).

(6) A transformant obtained by transformation of the recombinant vectorof (5).

(7) A method for assessing sensitivity of a rice blast fungus to ascytalone dehydratase inhibitor, comprising the steps of:

(a) identifying an amino acid in an amino acid sequence of scytalonedehydratase in a subject rice blast fungus, which corresponds to valineat position 75 in the amino acid sequence shown in SEQ ID NO: 4; and

(b) assessing sensitivity of the subject rice blast fungus to thescytalone dehydratase inhibitor based on the results of step (a).

(8) A method for assessing sensitivity according to (7), wherein whenthe amino acid identified in step (a) is methionine, the sensitivity ofthe subject rice blast fungus to the scytalone dehydratase inhibitor isassessed to be lower than that of a wild-type rice blast fungus in step(b).

(9) A kit for screening an inhibitor, comprising the scytalonedehydratase of (4).

(10) A kit for assessing a rice blast fungus resistant to a scytalonedehydratase inhibitor, comprising a pair of primers designed to flank anucleotide sequence coding for an amino acid corresponding to valine atposition 75 in the amino acid sequence shown in SEQ ID NO: 4.

(11) A kit for assessing a rice blast fungus resistant to a scytalonedehydratase inhibitor, comprising an oligonucleotide including anucleotide sequence coding for an amino acid corresponding to valine atposition 75 in the amino acid sequence shown in SEQ ID NO: 4.

Hereinafter, the present invention will be described in detail.

The gene according to the present invention codes for scytalonedehydratase (hereinafter, referred to as a “mutant SCDH enzyme”) thatexhibits scytalone dehydratase activity in the presence of a scytalonedehydratase inhibitor (hereinafter, referred to as an “SCDH inhibitor”).In the following description, scytalone dehydratase with decreasedscytalone dehydratase activity in the presence of an SCDH inhibitor issimply referred to as an “SCDH enzyme” or a “wild-type SCDH enzyme.”

Examples of the SCDH inhibitor include carpropamid(2,2-dichloro-N-(1-(4-chlorophenyl)ethyl)-1-ethyl-3-methylcyclopropanecarboxamide),fenoxanil(1-(2,4-dichlorophenyl)oxy-N-(1-cyano-1,2-dimethyl)propylethanecarboxamide),diclocymet(N-[1-(2,4-dichlorophenyl)ethyl]-1-cyano-2,2-dimethylpropanecarboxamide)and the like. The SCDH inhibitors are usually used as infectioninhibitors for rice with rice blast fungus to inhibit activity of theSCDH enzyme. Specifically, the SCDH enzyme catalyzes, in the melaninbiosynthesis pathway shown in FIG. 1, a dehydration reaction fromscytalone to 1,3,8-trihydroxynaphtalene (hereinafter, simply referred toas “1,3,8-THN”) and a dehydration reaction from vermelone to1,8-dihydroxynaphtalene.

The SCDH inhibitor inhibits the activity of this SCDH enzyme to preventformation of an appressorium in a rice blast fungus, thereby suppressingpathogenicity to rice. In other words, the SCDH inhibitor decreasesinfectivity of the rice blast fungus, and thus prevents an outbreak ofrice blast. A mutant SCDH enzyme, however, exhibits the above-describedenzyme activity even in the presence of the SCDH inhibitor and thusconfers resistance to the SCDH inhibitor upon rice blast fungi.Accordingly, a rice blast fungus that expresses a mutant SCDH enzyme(hereinafter, referred to as a “resistant rice blast fungus” or a“resistant strain”) does not allow inhibition of the melaninbiosynthesis even in the presence of an SCDH inhibitor, and anappressorium can be formed to rupture and invade a cuticular membrane ofthe a leaf surface. Thus, resistant rice blast fungi show highinfectivity even in the presence of the SCDH inhibitors.

Examples of the mutant SCDH enzyme include an enzyme having the aminoacid sequence shown in SEQ ID NO:2. The mutant SCDH enzyme may have anamino acid sequence similar to SEQ ID NO:2 with one or more amino acidsbeing deleted, substituted or added, which exhibits scytalonedehydratase activity in the presence of a scytalone dehydrataseinhibitor. As used herein, the expression “one or more” means, forexample, 1-30, preferably 1-20, and more preferably 1-10.

The enzyme activity of a wild-type SCDH enzyme or a mutant SCDH enzymecan be assessed by determining the dehydration reaction from scytaloneto 1,3,8-THN or the dehydration reaction from vermelone to1,8-dihydroxynaphtalene. Specifically, a reaction solution containingthe wild-type SCDH enzyme or the mutant SCDH enzyme and a substrate(scytalone or vermelone) is used to develop an enzyme reaction. Then, adecrease in the substrate and/or an increase in the reaction product(1,3,8-THN or 1,8-dihydroxynaphtalene) is determined, thereby assessingthe enzyme activity of the wild-type SCDH enzyme or the mutant SCDHenzyme.

Specifically, the enzyme reaction from scytalone to 1,3,8-THN may bedetermined spectroscopically. For example, the decrease in scytalone maybe determined according to Motoyama et al., J. Pestic. Sci, 23, 58-61,1998.

On the other hand, the increase in 1,3,8-THN may be determined by UVabsorption spectra of the scytalone substrate and the 1,3,8-THN productat 340-360 nm (as shown in FIG. 2). Although the absorption of scytaloneoverlaps with the absorption of 1,3,8-THN at 200-300 nm, the absorptionof scytalone at 340-360 nm is negligible. In the determination methodusing UV absorption spectra at 340-360 nm, a rate assay where the enzymereaction is determined for 100 seconds is employed to determine thesensitivity of the wild-type SCDH enzyme or the mutant SCDH enzyme tothe SCDH inhibitor.

According to this method, the enzyme reaction is proceeded in a reactionsolution, to which a predetermined concentration of an SCDH inhibitor(e.g., carpropamid) has been added, to determine UV absorption spectrumat 340-360 nm, thereby determining a synthesized amount of the reactionproduct, 1,3,8-THN. The determined synthesized amount of 1,3,8-THN isdivided by the synthesized amount of 1,3,8-THN in the absence of theSCDH inhibitor to obtain an inhibition rate of the SCDH inhibitor atthat concentration. The concentration of the SCDH inhibitor is varied todetermine the inhibition rates of the wild-type and mutant SCDH enzymesand calculate the I₅₀ value for each enzyme. From the I₅₀ value for thewild-type SCDH enzyme and the I₅₀ value for the mutant SCDH enzyme, R/Sratio is calculated to assess the sensitivity of the mutant SCDH enzymeto the SCDH inhibitor. For example, when the calculated R/S ratio is 2or higher, the mutant SCDH enzyme may be defined to have lowersensitivity to the SCDH inhibitor as compared to that of the wild-typeSCDH enzyme.

Determination of the enzyme activity of the mutant SCDH enzyme is notlimited to the above-described method, and any method may be applied.The method for determining enzyme activity of the mutant SCDH enzyme mayuse, for example, quantification of the enzyme reaction product,1,3,8-trihydroxynaphtalene, through HPLC analysis.

A gene coding for the mutant SCDH enzyme (hereinafter, referred to as a“mutant SCDH gene”) may be obtained from either genome DNA with intronsor cDNA without introns as long as it contains the nucleotide sequencecoding for the above-described mutant SCDH enzyme.

The mutant SCDH gene can be obtained by PCR using primers designed basedon the cDNA sequence of the SCDH enzyme from rice blast fungus andgenome DNA from a rice blast fungus resistant to the SCDH inhibitor(hereinafter, referred to as a “resistant rice blast fungus”). Themutant SCDH gene may also be obtained by RT-PCR using theabove-mentioned primers and mRNA extracted from the resistant rice blastfungus. The cDNA sequence of the SCDH enzyme from the rice blast fungusis known and described in Motoyama et al., Biosci. Biotech. Biochem, 62,564-566, 1988 (DNA databank, Accession no. AB004741).

Examples of the mutant SCDH gene obtained according to such methodsinclude the nucleotide sequence shown in SEQ ID NO: 1. The results ofcomparison between the nucleotide sequence (cDNA) of the mutant SCDHgene and that of a gene coding for wild-type SCDH enzyme (hereinafter,referred to as an “SCDH gene”) are shown in FIG. 3. The results ofcomparison between the nucleotide sequence of the mutant SCDH gene ingenome DNA and that of the SCDH gene are shown in FIG. 4. As shown inFIGS. 3 and 4, in the mutant SCDH gene, G (guanosine) at position 223 inthe SCDH gene is altered homozygously by A (adenosine). This alterationmeans that valine (Val) at position 75 in the wild-type SCDH enzyme ismutated into methionine (Met).

As a result of comparing the nucleotide sequence of the mutant SCDH genewith that of the SCDH gene, T (thymidine) at position 450 was found tobe mutated by C (cytidine) in the mutant SCDH gene. However, thisalteration does not result in amino acid mutation.

From comparisons in FIGS. 3 and 4, the mutant SCDH gene was found tohave an intron of 81 bases and an intron of approximate 89 bases betweenpositions 42 and 43 and positions 141 and 142 in the amino acid sequenceof the mutant SCDH enzyme, respectively. Since the latter intron(located between positions 141 and 142 in the amino acid sequence of themutant SCDH enzyme) was followed by poly(A) strand, and when PCR wascarried out, the resultant product had various lengths, exact lengththereof was unable to be determined. Therefore, it is expressed as“about 89 bases.”

The mutant SCDH gene is not limited to the nucleotide sequence shown inSEQ ID NO: 1, and may be any nucleotide sequence coding for a proteinconsisting of the amino acid sequence shown in SEQ ID NO: 2, or an aminoacid sequence shown in SEQ ID NO: 2 by deletion substitution or additionof one or more amino acids, which exhibits scytalone dehydrataseactivity in the presence of a scytalone dehydratase inhibitor. Examplesof such nucleotide sequence include a nucleotide sequence shown in SEQID NO: 1, which includes a nucleotide substitution that does not resultin amino acid mutation.

The mutant SCDH gene may be a nucleotide sequence coding for a proteinthat exhibits scytalone dehydratase activity in the presence of ascytalone dehydratase inhibitor, and capable of hybridizing to anucleotide sequence complementary to the nucleotide sequence shown inSEQ ID NO: 1 under stringent conditions. Stringent conditions mean, forexample, a sodium concentration of 10-300 mM, preferably 20-100 mM, anda temperature of 25-70° C., preferably 42-55° C.

The mutant SCDH gene may be obtained by PCR using, as a template, genomeDNA from a rice blast fungus that infects rice even in the presence ofthe SCDH inhibitor and a pair of primers with predetermined sequences.The genome DNA is prepared according to a method using CTBA(cetyltrimethylammonium bromide) as an extract solution, a method viaSDS/phenol or phenol/chloroform extraction, or with a commerciallyavailable kit (e.g., the DNeasy Plant System from Qiagen, the NucleonPhytoPure kit from Amersham Biosciences, etc.), although its preparationis not limited to these methods.

Furthermore, the mutant SCDH gene can be obtained by extracting totalmRNA from a rice blast fungus that infects rice even in the presence ofthe SCDH inhibitor and using the total mRNA and a pair of primers havingpredetermined sequences in RT-PCR. Total mRNA can be extracted from arice blast fungus, for example, by a guanidium method, an SDS-phenolmethod, phenol/chloroform extraction with the RNAeasy Total RNA Systemfrom Qiagen, the Quick Prep Micro mRNA Purification Kit or the QuickPrep Total RNA Extraction Kit from Amersham Biosciences, although itspreparation is not limited to these methods.

The pair of primers used in the above-described PCR and RT-PCR may bedesigned to flank the SCDH gene based on the nucleotide sequence ofgenome DNA from, for example, a rice blast fungus deposited with s genebank. The pair of primers may also be designed by further adding afunctional sequence based on a nucleotide sequence of genome DNA from arice blast fungus. Examples of functional sequences include a sequencerecognized by a restriction enzyme for linking to a vector, and aninsertion sequence for reading frame adjustment.

Examples of the pair of primers include, but are not limited to, thefollowing sequences: Primer 1 (SEQ ID NO: 5):5′-GCAGTGATACCCACACCAAAG-3′ Primer 2 (SEQ ID NO: 6):5′-TTATTTGTCGGCAAAGGTCTCC-3′ Primer 3 (SEQ ID NO: 7):5′-AGTTCGAACTGGAATTCAACCGGCACGCATGATGCATGCATTTA-3′ Primer 4 (SEQ ID NO:8): 5′-ATGGGTTCGCAAGTTCAAAAG-3′ Primer 5 (SEQ ID NO: 9):5′-GTGGCCCTTCATGGTGACGTCCT-3′ Primer 6 (SEQ ID NO: 10):5′-ACAAGCTCTGGGAGGCAATG-3′ Primer 7 (SEQ ID NO: 11):5′-ATCGTCGACGTGAATTCGTCTTGTAAAAGCCGCCAAC-3′

Primers 1, 4, 6 and 7 are sense primers while Primers 2, 3 and 5 areantisense primers. Therefore, one of the pair of primers is selectedfrom the sense primers and the other is selected from the antisenseprimers.

Primer 2 is synthesized based on the nucleotide sequence disclosed inpublication (Motoyama et al., Biosci. Biotech. Biochem, 62, 564-566,1988), and the underlined base is “G.” However, the corresponding basein Accession no. AB004741 from DNA data bank is “C.” Although thecorrect base is “C,” no effect is caused on the results from PCR andRT-PCR even when the base is “G.” Underlined letters in Primers 3 and 7indicate EcoRI recognized sequences. These EcoRI recognized sequencescan be exploited upon incorporation into a protein expression vector orthe like. Nucleotide sequences 5′ to the EcoRI recognized sequences inPrimers 3 and 7 are added to give enough margin for EcoRI to recognizethe EcoRI recognized sequences. In Primer 7, two nucleotide sequences 3′to the EcoRI recognized sequences (i.e., “GT” at positions 18 and 19 inPrimer 7) are nucleotides for allowing reading frame adjustment uponincorporation into a protein expression vector (pGEX-2T).

For example, RT-PCR is carried out using Primers 7 and 3 with total RNAas a template. The obtained PCR product is treated with EcoRI, and thenincorporated into pGEX-2T (Amersham Biosciences) that has been subjectedto EcoRI digestion and BAP treatment with alkaline phosphatase inadvance, thereby preparing a plasmid. The plasmid was deposited with theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki, Japan) on Mar. 8, 2002 under the Budapest Treaty, asRice Blast Mutant SCDH cDNA (FERM BP-7948).

This plasmid (Rice Blast Mutant SCDH cDNA) is capable of expressing anSCDH enzyme as a fusion protein with glutathione-S-transferase(hereinafter, referred to as “GST”) in a host such as E. coli. A plasmidwith a mutant SCDH gene may be constructed to be applicable to acell-free protein expressing system.

Furthermore, the mutant SCDH gene may be obtained using a predeterminedprobe and a cDNA library from rice blast fungi that infect rice even inthe presence of an SCDH inhibitor.

The mutant SCDH gene may also be obtained by mutagenesis of a wild-typeSCDH gene. For example, a mutant SCDH gene may be obtained through theso-called site-directed mutagenesis using primers designed to alter acodon in a wild-type SCDH gene coding for valine (Val) at position 75 bya codon coding for methionine (Met). A commercially available kit may beused to obtain a mutant SCDH gene using the site-directed mutagenesis.Examples of commercially available kits include the TaKaRa LA PCR invitro Mutagenesis kit (Takara).

The above-described mutant SCDH gene is useful for screening a novelSCDH inhibitor which decreases the infectivity of a resistant rice blastfungus, as illustrated in the Examples below. Specifically, anexpression vector operatively incorporating the above-described mutantSCDH gene is used to express the mutant SCDH enzyme, the enzyme activityof which is in turn determined in the presence of a candidate agent fora novel SCDH inhibitor. By determining whether or not the enzymeactivity of the mutant SCDH enzyme is decreased in the presence of thecandidate agent, a novel SCDH inhibitor can be screened.

Specifically, according to conventional determination methods,inhibition of appressorium formation by a rice blast fungus in thepresence of a candidate agent is assessed by a so-called pot test or atest based on observation of an appressorium involving the rupture ofcellophane affixed on an agar petri dish, and thus these methods arehardly capable of rapid screening for an SCDH inhibitor. On the otherhand, according to the above-described method, enzyme activity of anSCDH enzyme can be measured by a simple procedure, allowing rapidscreening for a novel SCDH inhibitor.

From the nucleotide sequence analysis of the above-described mutant SCDHgene, it was found that the mutant SCDH enzyme in which valine (Val) atposition 75 in the SCDH enzyme had been altered by methionine (Met)exhibited enzyme activity in the presence of the SCDH inhibitor.Therefore a nucleotide sequence coding for the amino acid at position 75in the SCDH enzyme may be analyzed to determine whether the subject SCDHgene has conferred resistance to an SCDH inhibitor.

Specifically, when investigating whether or not a rice blast fungus, forexample, from a predetermined region (a subject rice blast fungus) hassensitivity to an SCDH inhibitor, the amino acid at position 75 in theSCDH enzyme coded by the SCDH gene (the subject SCDH gene) from thesubject rice blast fungus may be identified to assess the sensitivity ofthe subject rice blast fungus to the SCDH inhibitor.

The nucleotide sequence coding for the amino acid at position 75 in thesubject SCDH enzyme may be identified according to any method and is notlimited to a particular method. In order to sequence the nucleotidesequence coding for the amino acid at position 75 in the SCDH enzyme,for example, at least a pair of primers designed to flank the nucleotidesequence comprising the nucleotide sequence coding for the amino acid atposition 75 in the SCDH enzyme and template DNA (cDNA or genome DNA) areused to sequence a predetermined region of the template DNA. Based onthe sequenced nucleotide sequence, the amino acid at position 75 in thesubject SCDH enzyme can be identified.

For sequencing the nucleotide sequence coding for the amino acid atposition 75 in the subject SCDH enzyme, the genome DNA as the templateis preferably obtained through solid cultivation of the subject riceblast fungus, followed by collection of filamentous mycelia andmicrowave irradiation of the mycelia. Irradiation with microwaves may becarried out, for example, using a microwave oven or the like. The genomeDNA as the template can be obtained in a short time by this method, ascompared to the standard method of harvesting the subject rice blastfungus after liquid culture and extracting genome DNA therefrom.

For determining the nucleotide sequence coding for the amino acid atposition 75 in the subject SCDH enzyme, one of the primers is preferablydesigned to hybridize near, for example, a location 40 bases upstreamfrom the nucleotide sequence coding for the amino acid at position 75.Consequently, the nucleotide sequence coding for the amino acid atposition 75 in the subject SCDH enzyme can be determined in a shorttime.

Furthermore, for determining the nucleotide sequence coding for theamino acid at position 75 in the subject SCDH enzyme, an oligonucleotidecomprising a nucleotide sequence coding for an amino acid correspondingto valine at position 75 in the amino acid sequence shown in SEQ ID NO:4 may be used. For example, the oligonucleotide is designed to hybridizeto the gene coding for the subject SCDH enzyme when the amino acid atposition 75 in the subject SCDH enzyme is methionine. Then, via colonyhybridization or Southern hybridization using this oligonucleotide as aprobe, the amino acid at position 75 in the subject rice blast fungusmay be identified. The sensitivity of the subject rice blast fungus toan SCDH inhibitor may also be assessed through this method.

Moreover, for analyzing the amino acid at position 75 in the subjectSCDH enzyme, single-stranded DNA conformation polymorphism (hereinafter,referred to as “SSCP”) may be exploited. Specifically, difference inmobility patterns between a wild-type SCDH gene and a resistance SCDHgene due to difference in single-stranded conformation is detected inadvance, and compared to a mobility pattern based on the single-strandedconformation of the subject SCDH gene. Accordingly, the nucleotidesequence of the subject SCDH gene coding for the amino acid at position75 in the SCDH enzyme can be identified. By exploiting SSCP foranalyzing the amino acid at position 75 in the subject SCDH enzyme,sensitivity of the subject rice blast fungus to the SCDH inhibitor canbe determined very quickly.

For analyzing the amino acid at position 75 in the subject SCDH enzyme,modified PCR-restriction fragment length polymorphism (RFLP) analysis(hereinafter, referred to as “modified PCR-RFLP method”) may also beapplied. Specifically, by modified PCR-RFLP analysis, mutation of valine(Val) at position 75 into methionine (Met) (hereinafter, referred to as“Val75Met mutation”) in the SCDH enzyme from the subject rice blastfungus can be tested in a simple manner.

In the modified PCR-RFLP analysis, one of the primers used for PCR doesnot comprise the base at position 223 (the base contained in the codoncoding for the amino acid at position 75 in the SCDH enzyme) and isdesigned to have a restriction-enzyme-recognized sequence at the 3′-enddepending upon the type of the base at position 223. This primer maycontain one or more bases partially mismatching the nucleotide sequenceof the genome DNA or cDNA as the template, while containing the aboverestriction-enzyme-recognized sequence. Therestriction-enzyme-recognized sequence is not particularly limited andmay be a sequence recognized by XbaI.

According to the modified PCR-RFLP analysis, first, PCR is performedusing a pair of primers designed as described above and genome DNA orcDNA as a template. Upon PCR, various conditions such as temperature ortime may appropriately be determined so that the desired region of thetemplate can be amplified even if a primer including one or more basesmismatching the template is used. The product resulting from PCRcontains a restriction-enzyme-recognized sequence as well as theabove-described primer depending on the base at position 223. Therestriction-enzyme-recognized sequence may not be contained depending onthe base at position 223.

Next, the product resulting from PCR is treated with a restrictionenzyme that recognizes the restriction-enzyme-recognized sequencecontained in the above-described primer The fragments obtained throughthis restriction enzyme treatment have different lengths due to thedifference of the base at position 223. Then, the lengths of thefragments obtained by the restriction enzyme treatment may be detected,for example, by a method such as electrophoresis to identify the base atposition 223 to analyze the amino acid at position 75 in the subjectSCDH enzyme.

Furthermore, for analyzing the amino acid at position 75 in the subjectSCDH enzyme, a generally known single nucleotide polymorphism typingmethod may be employed. Examples of the single nucleotide polymorphismtyping method include the SNaPshot Multiplex Kit from Applied Biosystems(single primer extension reaction), the Masscode system from Qiagen(mass spectrometry), the MassARRAY system from Sequenom, the UCAN methodfrom Takara, the Invader assay using Cleavase and a method using amicroarray.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a melanin biosynthesis pathway in arice blast fungus.

FIG. 2 is a characteristic diagram showing UV absorption spectra ofscytalone and 1,3,8-THN.

FIG. 3 shows comparison between the nucleotide sequence of the SCDH genefrom the rice blast fungus registered with the gene bank, a nucleotidesequence (cDNA) of an SCDH gene from a standard strain and a nucleotidesequence (cDNA) of an SCDH gene from a resistant strain.

FIG. 4 shows comparison between the nucleotide sequence of the SCDH genefrom the rice blast fungus registered with the gene bank, a nucleotidesequence (genome DNA) of an SCDH gene from a standard strain and anucleotide sequence (genome DNA) of an SCDH gene from a resistantstrain.

FIG. 5 is a characteristic diagram showing the relationship between thecarpropamid concentrations and the inhibition rates for SCDH enzymeactivity in crude enzyme solutions extracted from the standard strainand the resistant strains (A and B). In this diagram, the open circlesrepresent the results for the crude enzyme solution from the standardstrain, the open triangles represent the results for the crude enzymesolution from the resistant strain A and the open squares represent theresults for the crude enzyme solution from the resistant strain B.

FIG. 6 are electrophoresis pictures showing the results from thesingle-stranded DNA conformation polymorphism (SSCP) analysis conductedin Example 4, where A (left) shows the results from electrophoresiswithout purification using GFX PCR DNA and Gel Band Purification Kitwhile B (right) shows the results from electrophoresis followingpurification using GFX PCR DNA and Gel Band Purification Kit.

FIG. 7 is a schematic view showing a method for preparing plasmid RiceBlast wild SCDH cDNA and Rice Blast Mutant SCDH cDNA.

FIG. 8 is a characteristic diagram showing the relationship between thecarpropamid concentrations and the inhibition rates for SCDH enzymeactivity for the GST-fused SCDH enzyme obtained by expressing cDNA fromthe standard strain in E. coli, the GST-fused SCDH enzyme obtained byexpressing cDNA from the resistant strain in E. coli, the crude enzymesolution from the standard strain and the crude enzyme solution from theresistant strain. In this diagram, the open circles represent theresults for the crude enzyme solution from the standard strain, the opentriangles represent the results for the GST-fused SCDH enzyme expressedfrom the standard strain cDNA, the closed circles represent the resultsfor the crude enzyme solution from the resistant strain and the closedtriangles represent the results for the GST-fused SCDH enzyme expressedfrom the resistant strain cDNA.

FIG. 9 is a characteristic diagram showing the relationship between thefenoxanil or diclocymet concentrations and the inhibition rates for theGST-fused SCDH enzyme obtained by expressing cDNA from the standardstrain in E. coli and the GST-fused SCDH enzyme obtained by expressingcDNA from the resistant strain in E. coli. In this diagram, the opencircles represent the inhibition of the GST-fused SCDH enzyme expressedfrom the standard strain cDNA by fenoxanil, the open triangles representthe inhibition of the GST-fused SCDH enzyme expressed from the resistantstrain cDNA by fenoxanil, the closed circles represent the inhibition ofthe GST-fused SCDH enzyme expressed from the standard strain cDNA bydiclocymet, and the closed triangles represent the inhibition of theGST-fused SCDH enzyme expressed from the resistant strain cDNA bydiclocymet.

FIG. 10 is an electrophoresis (3% agarose gel) picture showing theresults obtained by analyzing Val75Met mutation in the SCDH enzyme byapplying PCR-RFLP method performed in Example 6.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLES

Hereinafter, the present invention will be described in more detail bymeans of examples. The technical scope of the present invention,however, is not limited by these examples.

Example 1

According to this example, first, filamentous mycelia used forextracting SCDH enzymes were prepared. Spore solutions (10⁵/ml)containing a rice blast fungus (Pyricularia oryzae) as a standard(wild-type) strain and carpropamid-resistant rice blast fungi (resistantstrains A and B) were individually added to 200 ml YGPCa liquid culturesolutions (pH 6.5) each containing yeast extract (5 g), glucose (20 g),KH₂PO₄ (0.5 g), Na₂HPO₄ (0.5 g) and CaCl₂ (0.5 mg), and were grown at27° C. for 4 to 5 days.

After the cultivation, the filamentous mycelia were collected throughcentrifugation of the culture solutions and washed with distilled water.Cold acetone, which has five times the weight of the mycelia, was added,and the results were homogenized with a Waring blender. The homogenateswere centrifuged (15,000×g, 20 min.). The precipitates were dried at 4°C. to obtain acetone powders, which were stored at −85° C.

The obtained acetone powders were used to prepare crude enzyme solutionscontaining the SCDH enzymes in order to determine their enzymeactivities. In order to prepare these crude enzyme solutions, each theacetone powder was suspended in 20 ml of 1/15 M potassium phosphatebuffer (pH 6.8) agitated for 30 minutes while being iced and thencentrifuged at 15,000×g for 15 minutes. Supernatants obtained bycentrifugation were used as the crude enzyme solutions.

Next, to determine the enzyme activities of the SCDH enzymes using thecrude enzyme solutions, first, 1,300 μl of 100 mM phosphate buffer (pH6.8) containing 1 mM EDTA, 30 μl of 20 mM scytalone (ethanol solution),30 μl ethanol solution of carpropamid at an appropriate concentrationand 1,440 μl ultrapure water were mixed and pre-incubated at 27° C. for2 minutes. Then, 200 μl of the crude enzyme solution was added toinitiate enzyme reaction. The amount of 1,3,8-THN produced fromscytalone through enzyme reaction was monitored for 100 seconds as anincrease in the absorbance at UV 350 nm, thereby determining enzymeactivity caused by the SCDH enzyme contained in the crude enzymesolution. The scytalone substrate was prepared from mycelium obtainedthrough liquid cultivation of the standard (wild-type) strain in thepresence of carpropamid, according to a routine technique (Kurahashi etal., J. Pestic. Sci, 23, 22-28, 1998).

The results are shown in FIG. 5. These results were used to calculate50% inhibition concentrations (I₅₀ values) by probit analysis. As aresult, the I₅₀ value of the crude enzyme solution extracted from thestandard (wild-type) strain with respect to carpropamid was 7.45 nMwhile those of the resistant strains A and B were 163 nM and 157 nM,respectively. From these values, the R/S ratio was about 21.5. Thissuggested that a factor for carpropamid resistance in the resistantstrains A and B was the decrease in the sensitivity of scytalonedehydratase, which is the target of the carpropamid.

Example 2

In this example, first, filamentous mycelia were prepared as describedbelow for extracting genome DNA and mRNA from rice blast fungi. First, astandard (wild-type) strain and carpropamid-resistant rice blast fungi(resistant strains A and B) were individually cultured on oatmeal media.After the cultivation, the mycelium parts were each added to 20 mlpotato-dextrose (PD) liquid media and pre-cultured at 28° C. for 3 days.Since the pre-cultured filamentous mycelia form themselves into lumps,they were homogenized with a sterilized Waring blender and 1 ml of eachsample was cultured in a 20 ml PD liquid medium for another 3-5 days.The mycelia were separated by filtration under reduced pressure andwashed with distilled water. These mycelia were ground in liquidnitrogen using a mortar. The ground powders were stored at −85° C. Thus,powders from the standard (wild-type) strain, the resistant strain A andthe resistant strain B were obtained.

For extracting total RNA using the powder from the resistant strain A,the Rneasy Plant Mini Kit (Qiagen) was used according to the attachedprotocol. For extracting genome DNA using the obtained powder, theDneasy Plant Mini Kit (Qiagen) was used according to the attachedprotocol. The RNA concentration was quantified by determining theabsorption at OD₂₆₀ with a spectrophotometer. DNA concentration wasdetermined by observation of the brightness on 1% agarose gel or bymeasurements of the fluorescence spectrum using Hoe 33258 (Hoechst).

Next, the obtained total RNA was used to prepare cDNA containing amutant SCDH gene from the resistant strain. In order to prepare cDNAcontaining the mutant SCDH gene, first, the obtained RNA (2 μg) wasmixed with 2 μl oligo(dT)₂₀ (10 pmol/μl), 2 μl each of Primer 1(5′-GCAGTGATACCCACACCAAAG-3′, 25 pmol/μl) and Primer 2(5′-TTATTTGTCGGCAAAGGTCTCC-3′, 25 pmol/μl) and RT-PCR beads (AmershamBiosciences) to a final volume of 50 μl to prepare a reaction solution.The reaction took place under the following conditions. For cDNAsynthesis, reaction was performed at 42° C. for 30 minutes, followed byreaction at 95° C. for 30 minutes. Subsequently, for PCR reaction usingthe synthesized cDNA as a template, 35 cycles of 95° C. for 30 seconds,55° C. for 1 minute and 72° C. for 1 minute were repeated. After thefinal cycle at 72° C. for 7 minutes, the reaction was carried out andterminated. The reaction solution obtained was purified after thereaction using the GFX PCR DNA and the Gel Band Purification Kit(Amersham Biosciences) to obtain the RT-PCR product. cDNA containing theSCDH gene from the standard strain and cDNA containing the mutant SCDHgene from the resistant strain B were also obtained in manners similarto the above-described method.

In addition, DNA containing the mutant SCDH gene from the resistantstrain A was prepared using the obtained genome DNA. For preparing thisDNA, first, 4 μl of the obtained genome DNA was mixed with 1 μl each ofPrimer 1 (5′-GCAGTGATACCCACACCAAAG-3′, 25 pmol/μl) and Primer 3(5′-AGTTCGAACTGGAATTCAACCGGCACGCATGATGCATGCATTTA-3′, 25 pmol/μl) and PCRbeads (Amersham Biosciences) to a final volume of 25 μl to prepare areaction solution. The reaction took place under the followingconditions. For PCR reaction using the genome DNA as a template, 40cycles of 95° C. for 30 seconds, 55° C. for 1 minute and 72° C. for 2minutes were repeated. After the final cycle at 72° C. for 7 minutes,the reaction was carried out and terminated. The reaction solutionobtained was purified after the reaction using the GFX PCR DNA and theGel Band Purification Kit (Amersham Biosciences) to obtain the PCRproduct. DNA containing the SCDH gene from the standard strain and DNAcontaining the mutant SCDH gene from the resistant strain B were alsoobtained in manners similar to the above-described method.

Then, the obtained RT-PCR product and the PCR product were used tosequence the nucleotide sequence of cDNA containing the mutant SCDH geneand the nucleotide sequence of DNA containing the mutant SCDH gene.Sequencing was performed using the BigDye Terminator Cycle Sequencing FSReady Reaction Kit from Applied Biosystems.

The sequencing reaction using this kit was performed in a reactionsolution (total amount: 20 μl) of a mixture of the RT-PCR or the PCRproduct as a template, 3.2 pmol primers (Primers 1, 3, 5 and 6) and 8 μlof terminator pre-mix. As the reaction conditions, 40 cycles of 96° C.for 10 seconds, 50° C. for 5 seconds and 60° C. for 4 minutes wererepeated. After the final cycle, the reaction was terminated at 60° C.for 7 minutes. After the reaction, the components such as the dieterminator remaining in the reaction solution were removed by gelfiltration using Auto Seq G-50 (Amersham Bioscience). Then, the reactionproduct was analyzed using ABI 310 Genetic Analyzer from AppliedBiosystems for nucleotide sequencing. The nucleotide sequence of themutant SCDH gene sequenced using the RT-PCR product as the template isshown in SEQ ID NO: 1 and the amino acid sequence of the mutant SCDHenzyme encoded by the mutant SCDH gene is shown in SEQ ID NO: 2.

The results from the analysis of cDNA of the mutant SCDH gene using theRT-PCR product as the template are shown in FIG. 3. FIG. 3 showscomparison between the nucleotide sequence of the SCDH gene from therice blast fungus registered with the gene bank (Accession no. AB004741,upper row), the nucleotide sequence of the SCDH gene analyzed using theRT-PCR product obtained from the standard strain (middle row) and thenucleotide sequence of the mutant SCDH gene analyzed using the RT-PCRproduct obtained from the resistant strain A (bottom row).

The results from analysis of the mutant SCDH gene present in the genomeDNA using the PCR product as the template are shown in FIG. 4. FIG. 4shows comparison between the nucleotide sequence of the SCDH gene fromthe rice blast fungus registered with the gene bank (Accession no.AB004741, upper row), the nucleotide sequence of the SCDH gene analyzedusing the PCR product obtained from the standard strain (middle row) andthe nucleotide sequence of the mutant SCDH gene analyzed using the PCRproduct obtained from the resistant strain A (bottom row).

Referring to FIGS. 3 and 4, G (guanosine) at position 223 in the cDNAnucleotide sequence of the SCDH gene was found to have alteredhomozygously by A (adenosine) in the resistant strain A. This means thatvaline (Val) at position 75 in the amino acid sequence of the SCDHenzyme from the standard strain will be mutated into methionine (Met).The base at position 450 in the cDNA nucleotide sequence was T(thymidine) in the registered nucleotide sequence (Accession no.AB004741, upper row in FIG. 3) while it was C (cytidine) in the standardstrain and the resistant strain. However, since the alteration of thebase at position 450 in these cDNA nucleotide sequences is notassociated with amino acid mutation, it presumably has nothing to dowith sensitivity to SCDH inhibitors.

From FIG. 4, introns with lengths of 81 bases and about 89 bases wereconfirmed between positions 42 and 43 and positions 141 and 142,respectively, in the nucleotide sequence shown in SEQ ID NO: 3. Sincethe latter intron was followed by poly(A) strand and the productsresulting from PCR had various lengths, the exact length thereof wasunable to be determined. Accordingly, it is expressed as “about 89bases.”

Example 3

A simple assay of mutation of valine (Val) into methionine (Met) atposition 75 (hereinafter, referred to as “Val75Met mutation”) in theSCDH enzymes from rice blast fungi was considered.

A rice blast fungus grown on an oatmeal medium (5% oatmeal 2% sucroseand 1.5% agar) at 28° C. was pricked with a toothpick and transferredinto a 1.5 μl microtube. The microtube was covered with a lid andirradiated with microwave in a microwave oven (600 W) for 5-7 minutes.Due to this treatment, the cell wall of the fungus was ruptured.

Next, 50 μl TE buffer (pH 8.0) was added to the microtube, and theresultant was thoroughly agitated and centrifuged at 14,000 rpm for 10minutes. The supernatant containing free genome DNA was transferred toanother microtube and stored at −20° C. One to five μl of thesupernatant was mixed with 1 μl each of Primer 4(5′-ATGGGTTCGCAAGTTCAAAAG-3′, 25 pmol/μl), Primer 5(5′-GTGGCCCTTCATGGTGACCTCCT-3′, 25 pmol/μl) and PCR beads (AmershamBiosciences) for a final volume of 25 μl to prepare a reaction solution.For PCR reaction, 40 cycles of 95° C. for 30 seconds, 55° C. for 1minute and 72° C. for 2 minutes were repeated. After the final cycle at72° C. for 7 minutes, the reaction was carried out and terminated. Thereaction solution was purified using the Invisorb Spin PCRapid Kit(Invitek) to obtain a PCR product. The PCR product contained in thereaction solution was subjected to sequencing reaction using the BigDyeTerminator Cycle Sequencing FS Ready Reaction Kit from AppliedBiosystems.

For the sequencing reaction, the PCR product as a template, 3.2 pmol ofPrimer 6 (5′-ACAAGCTCTGGGAGGCAATG-3′) and 8 μl of terminator pre-mixwere mixed to prepare a reaction solution for a total amount of 20 μl.For the sequencing reaction, 40 cycles of 96° C. for 10 seconds, 50° C.for 5 seconds and 60° C. for 4 minutes were repeated. After the finalcycle at 60° C. for 7 minutes, the reaction was carried out andterminated. After the reaction, components such as the die terminatorremaining in the reaction solution were removed by gel filtration usingthe Auto Seq G-50 (Amersham Bioscience). Then, the reaction product wassubjected to sequence analysis using the ABI 310 Genetic Analyzer fromAmersham Biosciences. By using a 47 cm×50 μm short capillary column fromAmersham Biosciences, mutation of the amino acid valine at position 75into methionine was confirmed in a short time of about 35 minutes persample.

Example 4

A simple assay of mutation of valine (Val) into methionine (Met) atposition 75 (hereinafter, referred to as Val75Met mutation) in an SCDHenzyme from a rice blast fungus was considered by applying asingle-stranded DNA conformation polymorphism (SSCP) analysis.

As in Example 3, a genome DNA solution was simply prepared byirradiating rice blast fungus filamentous mycelium with microwaves. Fiveμl of this genome DNA solution were mixed with 1 μl each of Primer 6(5′-ACAAGCTCTGGGAGGCAATG-3′, 25 pmol/μl), Primer 5(5′-GTGGCCCTTCATGGTGACCTCCT-3′, 25 pmol/μl) and PCR bead (AmershamBiosciences) for a final volume of 25 μl to prepare a reaction solution.For PCR reaction, 40 cycles of 95° C. for 30 seconds, 55° C. for 1minute and 72° C. for 2 minutes were repeated. After the final cycle,the reaction was terminated at 72° C. for 7 minutes. As a result of thisreaction, 215 bp PCR product was obtained. The components such as taqDNA polymerase and primers remaining in the reaction solution wereremoved using GFX PCR DNA and the Gel Band Purification Kit (AmershamBiosciences).

Thereafter, a mixture of 0.4 ml of 0.5 M EDTA (pH 8.0), 10 mg ofbromophenol blue and 10 ml of formamide was prepared as a loading bufferfor SSCP. The reaction solution and the loading buffer were mixed at aratio of 1:1, heated at 85° C. for 15 minutes and cooled at once. As aresult, the PCR product contained in the reaction solution becamesingle-stranded DNA.

Then, the mixture of the reaction solution and the loading buffer wereused to perform electrophoresis with the PhastSystem full automaticelectrophoresis system from Amersham Biosciences. PhastGel Homogeneous12.5 and PhastGel Native Buffer Strips from Amersham Biosciences wereused as a gel carrier and a buffer reagent, respectively, forpre-electrophoresis at 400 V, 10 mA, 2.5 W, 4° C., and 100 Vh and foractual electrophoresis at 400 V, 10 mA, 2.5 W, 4° C., and 200 Vh. Theresults are shown in FIGS. 6A and 6B. FIG. 6A shows the results fromelectrophoresis without the above-described purification using GFX PCRDNA and the Gel Band Purification Kit. FIG. 6B shows the results fromelectrophoresis following the above-described purification using GFX PCRDNA and the Gel Band Purification Kit.

The electrophoresis patterns of the single stranded DNA are different inFIGS. 6A and 6B, presumably due to buffer compositions in the PCRsolutions. In any case, difference in the electrophoresis patternsbetween the standard strain and the carpropamid-resistant strains wasobserved and distinguishable from FIGS. 6A and 6B.

Example 5

An expression vector incorporating the mutant SCDH gene was constructedto study its resistance to an SCDH inhibitor.

In order to incorporate a scytalone dehydratase gene from a rice blastfungus into a protein expression vector pGEX-2T (Amersham Biosciences),RT-PCR was conducted using Primer 7(5′-ATCGTCGACGTGAATTCGTCTTGTAAAAGCCGCCAAC-3′) and Primer 3(5′-AGTTCGAACTGGAATTCAACCGGCACGCATGATGCATGCATTTA-3′) having EcoRIcleavage sites at their terminals. The RT-PCR was conducted according tothe method described in Example 1. Primers 7 and 3 were located upstreamand downstream from the open reading frame (ORF) of the SCDH gene,respectively, so as to flank the whole coding region for the SCDHenzyme.

For RT-PCR, first, total RNA (2 μg each) extracted from the standard(wild-type) fungus or the carpropamid-resistant rice blast fungus weremixed with 2 μl oligo(dT)₂₀ (10 pmol/μ), 2 μl each of Primer 4 (25pmol/μl) and Primer 3 (25 pmol/μl) and RT-PCR bead (AmershamBiosciences) to prepare a reaction solution for a final volume of 50 μl.The reaction took place under the following conditions. For cDNA strandsynthesis, the reaction solutions were reacted at 42° C. for 30 minutes,followed by reaction at 95° C. for 30 minutes. Subsequently, PCRreaction was performed by repeating 25 cycles of 95° C. for 30 seconds,55° C. for 1 minute and 72° C. for 1 minute. After the reaction, RT-PCRproducts were purified from the reaction solutions using GFX PCR DNA andthe Gel Band Purification Kit (Amersham Biosciences) and then elutedwith a final volume of 50 μl of sterilized water.

Next, 30 μl of the solution containing one of the RT-PCR products wasmixed with 4 μl of 10× H buffer (Takara), 1 μl of EcoRI (12 u/μl,Takara) for a final volume of 40 μl and subjected to restriction enzymereaction at 37° C. for 2 hours. After the restriction enzyme reaction,the reaction solutions were purified with GFX PCR DNA and the Gel BandPurification Kit (Amersham Biosciences) and eluted with 30 μl sterilizedwater.

In addition, 1 μg of GST-fused protein expression vector pGEX-2T(Amersham Biosciences) was mixed with 1 μl of 10× H buffer (Takara) and1 μl of EcoRI (12 u/μl, Takara) for a final volume of 10 μl andsubjected to a restriction enzyme reaction at 37° C. for 1 hour. To thisreaction Solution, 10 μl of BAP buffer (TOYOBO), 2.5 μl of alkalinephosphatase (0.4 u/μl, BAP-101, TOYOBO) and 77.5 μl of sterilized waterwere added. The resultant was subjected to dephosphorylation reaction at37° C. for 2 hours.

Then, reaction solutions were prepared by mixing 2 μl of theEcoRI-digested RT-PCR product, 1 μl of EcoRI/BAP-treated pGEX-2T, 2 μlsterilized water and 5 μl ligation buffer 1 (Ver. 2, Takara) andsubjected to ligation reaction at 16° C. for 12 hours. After thereaction, by following the protocol attached to the competent cell of E.coli (strain JM109) (Takara), the reaction solutions were used totransform E. coli JM109. Then, the transformed E. coli JM109 were spreadover LB solid media each containing 50 ppm ampicillin and subjected tostatic culture at 37° C. for 12 hours. After the cultivation, a fewsingle colonies were scraped to perform direct colony PCR. As a resultof the direct colony PCR, pGEX-2T inserted with the SCDH gene in thedirection of interest were screened. The nucleotide sequence was furthersequenced to confirm that the nucleotide sequence of the inserted SCDHgene was correct. This method is schematically illustrated in FIG. 7.The plasmid obtained according to this method was deposited with theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki, Japan) on Mar. 8, 2002 under the Budapest Treaty, asRice Blast Mutant SCDH cDNA (FERM BP-7948).

Then, E. coli transformed with a pGEX-2T vector containing the correctlyinserted SCDH gene was cultured at 27° C. in 200 ml LB liquid mediumcontaining 50 ppm ampicillin until OD₂₆₀ became 0.6-1.0. Thereafter,isopropyl-1-thio-β-D-galactoside (IPTG) was added to a finalconcentration of 1 mM and further subjected to thorough agitationculture at 27 C for 5 hours. After the cultivation, E. coli wascollected by centrifugation (10,000×g, 10 minutes, 4° C.). E. coli wasonce suspended in 10 ml of cold 1/15 M potassium phosphate buffer (pH6.8) for washing, and then collected by another centrifugation(10,000×g, 10 minutes, 4° C.). Subsequently, E. coli was again suspendedin 5 ml of cold 1/15 M potassium phosphate buffer (pH 6.8), subjected toultrasonic treatment using a microchip while icing, and centrifuged at4° C., 15,000×g for 20 minutes. The supernatants were used as crudeenzyme solutions.

The crude enzyme solutions were used to determine sensitivity tocarpropamid. The sensitivity to carpropamid was determined in the samemanner as described in Example 1. The results are shown in FIG. 8. InFIG. 8, the open circles and closed circles represent the results fromdetermination of sensitivity to carpropamid measured in Example 1.

Referring to FIG. 8, for both the standard fungus andcarpropamid-resistant fungi, the GST-fused SCDH enzymes expressed in E.coli exhibited the same drug sensitivity as the SCDH enzyme contained inthe crude enzyme solutions extracted from rice blast fungi.

Similarly, sensitivity to SCDH inhibitors, fenoxanil and diclocymet,were also studied. The results are shown in FIG. 9.

Referring to FIG. 9, the GST-fused SCDH enzyme was also found to showresistance to fenoxanil and diclocymet. In other words, the resultsshown in FIGS. 8 and 9 revealed that the GST-fused SCDH enzyme showedhigh enzyme activity in the presence of various SCDH inhibitors.Accordingly, in order to find and/or develop drugs for preventing anddisinfestating for rice blast fungi that exhibit high infectivity torice even in the presence of SCDH inhibitors, candidate agents may bescreened using the GST-fused SCDH enzyme. Specifically, the enzymeactivity of the GST-fused SCDH enzyme is measured in the presence ofcandidate agents to select a candidate agent that significantlydecreases the enzyme activity. The selected candidate agent decreasesthe enzyme activity of the mutant SCDH enzyme and thus decreases theinfectivity of the resistant rice blast fungus. Accordingly, developmentof rice blast caused by resistant rice blast fungi can be prevented.

Example 6

A simple assay for Val75Met mutation in an SCDH enzyme from a rice blastfungus was considered by applying the PCR-RFLP method. Similar toExample 3, a rice blast fungus filamentous mycelium was irradiated withmicrowaves to simply prepare a genome DNA solution. Five μl of thisgenome DNA solution was mixed with 1 μl each of Primer 8 (SEQ ID NO: 12,5′-TTCGTCGGCATGGTCTCGAGCATCTAG-3′, 25 pmol/μl), Primer 5(5′-GTGGCCCTTCATGGTGACCTCCT-3′, 25 pmol/μl) and PCR bead (AmershamBioscience) for a final volume of 25 μl to prepare a reaction solution.

The underlined bases “TCT” in Primer 8 mismatch the nucleotide sequenceof the genome DNA as a template and are designed to form a cleavagerecognized site (“TCTAGA”) for restriction enzyme XbaI together with thebases “AG” at the 3′-end and the first base that is amplified by thelater-described PCR. When the first base amplified by the PCR is “A,”the fragment amplified by Primer 8 will include the cleavage recognizedsite for restriction enzyme XbaI. On the other hand, when the first baseamplified by the PCR is a base other than “A,” the cleavage recognizedsite for restriction enzyme XbaI is absent in the amplified fragment.

For PCR reaction, 40 cycles of 95° C. for 30 seconds, 55° C. for 1minute and 72° C. for 2 minute were repeated. After the final cycle at72° C. for 7 minutes, the reaction was carried out and terminated. As aresult of this reaction, a PCR product of 183 bp was obtained. The PCRproduct was purified using GFX PCR DNA and the Gel Band Purification Kit(Amersham Biosciences) and then eluted with a final volume of 20 μl ofsterilized water. Of the resultant, 7.5 μl was mixed with 1 μl 10× Mbuffer (Takara), 1 μl 0.1% BSA solution and 0.5 μl XbaI (12 u/μl,Takara) for a final volume of 10 μl and subjected to restriction enzymereaction at 37° C. for 1 hour. Results from electrophoresis of the totalvolume of the reaction solution in 3% agarose are shown in FIG. 10. InFIG. 10, Lane 2 represents the reaction solution using the genome DNAextracted from the standard strain. Lane 3 represents the reactionsolution using the genome DNA extracted from the resistant strain. Lane4 represents the reaction solution using the genome DNA extracted fromthe standard strain, which had not been subjected to restriction enzymereaction. Lane 5 represents the reaction solution using the genome DNAextracted from the resistant strain, which had not been subjected torestriction enzyme reaction.

As can be appreciated from FIG. 10, the XbaI-treated sample of the PCRproduct from the resistant strain was shorter by about 25 bases. Fromthis result, it became clear that the standard strain (wild-type strain)and the resistant strain can be distinguished by applying the PCR-RFLPtechnique.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides a gene that can beused extensively, for example, in studies relating to rice blast fungiresistant to SCDH inhibitors. This gene may be used, for example, inscreening a novel SCDH inhibitor and assessing sensitivity of a subjectrice blast fungus to an SCDH inhibitor.

Free Text in Sequence Listing

SEQ ID NOS: 5-12 are synthesized primers.

1. A gene coding for either one of the following proteins (a) or (b):(a) a protein consisting of the amino acid sequence shown in SEQ IDNO:2; or (b) a protein consisting of an amino acid sequence shown in SEQID NO:2 by deletion substitution or addition of one or more amino acids,which exhibits scytalone dehydratase activity in the presence of ascytalone dehydratase inhibitor.
 2. A gene according to claim 1, whereinthe scytalone dehydratase inhibitor inhibits dehydration reaction fromscytalone to 1,3,8-trihydroxynaphtalene in a melanin biosynthesispathway.
 3. A gene according to claim 1, wherein the scytalonedehydratase inhibitor is carpropamid.
 4. A scytalone dehydratase encodedby the gene of claim
 1. 5. A recombinant vector comprising the gene ofclaim
 1. 6. A transformant obtained by transformation of the recombinantvector of claim
 5. 7. A method for assessing sensitivity of a rice blastfungus to a scytalone dehydratase inhibitor, comprising the steps of:(a) identifying an amino acid in an amino acid sequence of scytalonedehydratase in a subject rice blast fungus, which corresponds to valineat position 75 in the amino acid sequence shown in SEQ ID NO: 4; and (b)assessing sensitivity of the subject rice blast fungus to the scytalonedehydratase inhibitor based on the results of step (a).
 8. A method forassessing sensitivity according to claim 7, wherein when the amino acididentified in step (a) is methionine, the sensitivity of the subjectrice blast fungus to the scytalone dehydratase inhibitor is assessed tobe lower than that of a wild-type rice blast fungus in step (b).
 9. Akit for screening an inhibitor, comprising the scytalone dehydratase ofclaim
 4. 10. A kit for assessing a rice blast fungus resistant to ascytalone dehydratase inhibitor, comprising a pair of primers designedto flank a nucleotide sequence coding for an amino acid corresponding tovaline at position 75 in the amino acid sequence shown in SEQ ID NO: 4.11. A kit for assessing a rice blast fungus resistant to a scytalonedehydratase inhibitor, comprising an oligonucleotide including anucleotide sequence coding for an amino acid corresponding to valine atposition 75 in the amino acid sequence shown in SEQ ID NO: 4.